As a leading cause of hospital acquired infection, enterococci account for nearly 10 % of all nosocomial infections. In addition, enterococci are a leading cause of sub-acute endocarditis in the community resulting in significant morbidity and mortality. Furthermore these infections pose a significant treatment challenge due to the presence of multi-drug resistance. The goal of this study is to investigate the underlying mechanisms of how the coregulated Enterococcus faecalis proteases, gelatinase (GelE) and the serine protease (SprE) contribute to the development of biofilms through the regulation of autolytic processes. The major autolysin, AtlA, is a target of both proteases and plays a key role in the development of biofilms by its ability to hydrolyze the cell wall leading to the death of sub-populations of bacteria and subsequent release of extracellular DNA. The DNA released from lysed cells serves as an important biofilm matrix upon which enterococcal biofilms develop. We have recently shown that this process is governed by a fratricidal mechanism, whose function is controlled by the Fsr quorum system. The interaction of AtlA with both proteases, as well as its association with the cell wall will be examined by constructing isogenic mutants of AtlA that lack specific LysM modular domains, as well as mutants that lack the N-terminal T/E rich domain. These mutants will be assessed for their autolytic capacity and contribution to biofilm formation. The interaction of AtlA and its mutants with both proteases will also be assessed using purified proteins in surface plasmon resonance studies to determine binding affinities, and the localization of native and mutant forms of AtlA to the cell surface will be examined by immunoelectron microscopy. In addition, modifications to the cell wall thought to modulate the autolytic activity of AtlA will be explored by generating and examining mutants defective for O-acetyl transferase activity, as well as D-alanylation of teichoic acids in autolysis and biofilm formation. Lastly, to determine the extent to which DNA-dependent biofilm processes contribute to disease in vivo, the relative contribution of the proteins involved in the regulation of autolysis in E. faecalis will be examined by comparing mutants in the catheter- induced rabbit model of infectious endocarditis.